Isolation and Purification of Tetrahymena nuclei
The procedures described below allow the isolation of nuclei from all stages of the Tetrahymena life cycle in high yield with a high degree of purity. This method gives highly purified populations of both micronuclei and macronuclei. Materials *Medium A (0.1 M Sucrose, 2 mM MgCl2, 4% Gum Arabic, 10 mM Tris pH 7.5, 1 mM Iodoacetamide, 10 mM Butyric Acid) *Medium B, freshly prepared (Medium A, Octanol, 100 mM PMSF) *Methyl Green (6 mL Glacial Acetic Acid, 29 mg CaCl2, 0.4 g methyl green) *Nucleus Wash Buffer (0.25 M Sucrose, 10 mM Tris, 3 mM CaCl2, 1 mM MgCl2, 1 mM Iodoacetamide, 10 mM Butyric Acid) *Octanol *PMSF (100 mM) *''Tetrahymena'' cell culture *Blender, prechilled to 4°C *Hemocytometer *Conical centrifuge bottles, 250 mL Method For vegetatively grown cells, add octanol to a final concentration of 0.63% (6.3 mL/L). For starved or mated cells, add octanol to medium A so that the final concentration of octanol is 0.15% in medium B. Cells that are already frozen in medium B do not require additional octanol. In the latter case, add medium A plus the appropriate amount of PMSF in a volume that is 3 times the volume of the frozen cells. Octanol helps to form the skin above the supernatant that holds micronuclei and cell debris, preventing them from being pelleted during initial rounds of centrifugation. Too much octanol interferes with nuclear pelleting; in contrast, too little octanol causes nuclei to pellet more easily, but allows increased contamination of the nuclear fractions with cell debris. #Determine the number of cells to be used in the preparation, and prepare appropriate sized culture of Tetrahymena. Typically a 1-2 L culture (2.5-5 x10^5 cells/mL) provides a useful size for starters. #Just prior to harvesting the cells, add protease inhibitors (typically, add PMSF to a final concentration of 1 mM to all buffers) to all solutions while stirring vigorously in the cold. All solutions and procedures are performed at 4°C or on ice to minimize proteolysis. #Add octanol to medium A only, and mix well (see below for required concentration). The addition of octanol and PMSF to medium A converts medium A to medium B.'' #Harvest cells by centrifuging in 250-ml conical centrifuge bottles at 1500-2000g at 2°C for 5 minutes in a swinging-bucket rotor. #Quickly decant the supernatant and vortex to loosen the pellet. #Resuspend the pelleted cells directly in medium B. For vegetatively growing cells, resuspend to a final concentration of 1.5 x 10^6 cells/mL of medium B. For starved or mating cells, resuspend to a final concentration of 1 x 10^5 to 5 x 10^5 cells/mL of medium B. '''''Cell Disruption #Blend to disrupt cells in a cold blender for 30-60 seconds on high speed. #Check the homogenate to ensure that micronuclei have been removed from their “cup” next to the macronuclei by placing a small portion on a slide, adding methyl green (~1:1), and inspecting using a light microscope. If less than 10% of micronuclei are still attached to macronuclei, begin differential nuclear pelleting; if not, reblend and check again. ''Differential Nuclear Pelleting #Pour blended cells into conical centrifuge bottles and spin at 1500-2000g at 0-4 C for 5 minutes in a swinging-bucket rotor. ''Volumes of 50-250 mL/centrifuge bottle can be used. #Gently shake the bottles to loosen the skin that forms on top of the supernatant, and decant the supernatant and skin into the cold blender. #Resuspend pellets in a small amount of nucleus wash buffer (5 mL) and examine a small aliquot under the light microscope after staining with methyl green (1:1). Determine the relative numbers of macronuclei and micronuclei. #Collect the pellet into a tube (15-50 mL conical centrifuge tube) marked Pn (where n = 1 for 1st pellet, 2 for 2nd pellet, etc.) #Do not pool pellets from different rounds of centrifugation until you have analyzed aliquots of all the pellets using the microscope. #Blend the supernatant for 20 seconds on high speed and centrifuge at a somewhat higher speed (2500-3000g) for 5 minutes. The speed is somewhat dependent on the number of macronuclei and micronuclei seen in the previous pellet fraction; if lots of macronuclei and no micronuclei are seen, increasing the time or speed of centrifugation will usually help pellet micronuclei. #Collect the supernatant and resuspend the pellet (P2) in nucleus wash buffer and evaluate the nuclei as in Step 11. #Repeat blendings and centrifugations, increasing the speed of the centrifugations and analyzing each pellet (Steps 13-14), until there are very few macronuclei in the pellet fraction. As the number and the speed of centrifugation increase, the number of micronuclei in the pellet will increase and the number of macronuclei in the pellet will eventually decrease. #When the majority of nuclei in the last observed pellet are micronuclei, blend the supernatant for 20 seconds on high speed and centrifuge in any swinging-bucket rotor at about 5000g at 0-4 C for 5 minutes. #Collect the supernatant, suspend the pellet (Pn) in nucleus wash buffer, and evaluate the pellet (Step 11). ''Count Total Number of Nuclei Isolated #Pool appropriate pellets containing macronuclei; separately, do the same for micronuclei. Resuspend nuclei in convenient volumes (e.g., 50 ml for 1 liter of mid-log growth culture) using nucleus wash buffer. #Dilute a small aliquot (5-10 ml) of pooled macronuclei or micronuclei (1:1) with methyl green. Count the nuclei on a hemocytometer and calculate the percent yield based on the number of starting cells. Assume one micro- and one macronucleus/cell during growth or starvation; numbers of nuclei/cell vary considerably during conjugation, depending on the stage. #When satisfied with the yield of macronuclei and/or micronuclei, harvest the nuclei by centrifuging at 2000g at 0-4 C for 5 minutes. ''If the micronuclear yield is low, repeat the blending and centrifugation Steps 8 and 9 (at 5000g) using a longer time. The homogenate (supernatant plus skin) can also be diluted with more medium A to which PMSF has been added, blended again, and then centrifuged at 5000g. #Wash the nuclei by resuspending them in a small amount of nucleus wash buffer and centrifuging them in microfuge tubes for 5 minutes. #Nuclei can be used immediately or stored in aliquots as nuclear suspensions in nucleus wash buffer at −80 C. ''Troubleshooting''